1. Field of the Invention
The present invention relates to the field of immunology, and more particularly to immunomodulation. The present invention provides novel compounds and methods for immunological intervention in disease states by employing synthetic polysaccharide antigens (SPAs) possessing immunomodulatory properties. These SPAs can be used in humans and other animals to provide protection against and/or treatment of inflammatory pathologies, or to induce a controlled inflammatory response to treat disease states or conditions in which an inflammatory response is therapeutically beneficial, for example in antiviral therapy, anticancer therapy, or as a vaccine adjuvant.
2. Description of Related Art
Microbial antigens are the most powerful immunomodulators known. Among the most common examples are lipopolysaccharide (LPS) from Gram negative bacteria, and bacterial cell wall glycopeptides, also known as murein or peptidoglycan (PG), from both Gram negative and Gram positive bacteria. Bacterial PG is well established as a potent inflammatory agent (Wahl et al. (1986) J. Exp. Med. 165:884).
Many microbial antigens, including PG, are thought to exert their pro-inflammatory effects by activating one of the mammalian cell surface receptors known as Toll-like receptors (TLRs). Activation of a TLR triggers an intracellular signaling pathway that leads to the induction of the transcription factor NF-κB, which in turn induces expression of genes encoding inflammatory mediators (chemokines and certain cytokines). PG itself is thought to activate through TLR2 (Takeuchi et al. (1999) Immunity 11:443).
Recently, cDNA array technology has brought even higher resolution to our understanding of pro-inflammatory mediator induction by PG (Wang et al. (2000) J. Biol. Chem. 275:20260). The most highly activated genes are those expressing chemokines (IL-8 and MIP-1β), and the second most highly activated genes are those expressing cytokines (TNF-α, IL1, and IL6). Regardless of mechanistic detail, the downstream effect of bacterial PG on the host is a potent inflammatory response. In fact, PG has long been used for induction of arthritis in animal models (Cromartie et al. (1977) J. Exp. Med. 146:1585). Partially purified PG from the bacterium Streptococcus pyogenes is now commercially available for such purpose (Lee Laboratories, Atlanta, Ga.).
Low molecular weight fragments of PG, known collectively as muropeptides, also exhibit inflammatory effects in animals, and these effects are dependent on muropeptide structure (Tuomanen et al. (1993) J. Clin. Invest. 92:297). Even the very smallest fragments of PG, designated muramyl dipeptide (MDP), and glucosaminyl MDP (GMDP), as well as their derivatives, exhibit inflammatory effects in animals (Kohashi et al. (1980) Infect. Immun. 29:70). While the high molecular weight PG induces pro-inflammatory responses through cell surface located TLR2, low molecular weight fragments of PG induce their pro-inflammatory activities through intracellular receptors known as Nod1 and Nod2 (Girardin et al., published on the web on Jul. 18, 2003 in J. Biol. Chem. as manuscript M307198200).
Kasper and Tzianabos have demonstrated that certain polysaccharides purified from the surface of bacterial cells exhibit protective effects in vivo when tested in models of inflammation such as the formation of intraabdominal abscesses, intraabdominal sepsis, and post-surgical adhesions (U.S. Pat. Nos. 5,679,654 and 5,700,787; PCT International Publications WO 96/07427, WO 00/59515, and WO 02/45708). These investigators have demonstrated that when purified from whole capsule, certain polysaccharides derived from Bacteroides fragilis, Staphylococcus aureus, and Streptococcus pneumoniae have unique characteristics that set them apart from many polysaccharide antigens. The former molecules are high molecular weight, helical, and zwitterionic in nature (Wang et al. (2000) Proc. Natl. Acad. Sci. USA 97:13478-13481, and references 5-9 therein). Most bacterial polysaccharides are neutral or negatively charged, and are considered to be T cell-independent antigens (Abbas et al. (2000) Cellular and Molecular Immunobiology, W.B. Saunders, Philadelphia). Kasper and Tzianabos suggest that the zwitterionic nature of these polysaccharides plays a role in their interaction with CD4+ T cells (Tzianabos et al. (1993) Science 262: 416-419; Tzianabos et al. (2001) Proc. Natl. Acad. Sci. USA 98:9365-9370). More recent work by this group suggests that some of these molecules may interact with antigen presenting cells (APCs) via their zwitterionic characteristics and further, that stimulation of CD4+ T cells by these polysaccharide antigens is dependent on MHC II-bearing APCs (Kalka-Moll et al. (2002) J. Immunol. 169:6149-6153). It has yet to be determined precisely how these interactions between zwitterionic polysaccharides and APCs may stimulate CD4+ T cells. These investigators have shown that zwitterionic polysaccharides activate CD4+ T cells in vitro as evidenced by the stimulation of proliferation and the production of the cytokines IL2, INFγ, and IL10, and that the protection is adoptively transferred by polysaccharide-stimulated T cells in vivo (PCT International Publication WO 00/59515; Kalka-Moll et al. (2000) J. Immunol. 164:719-724; Tzianabos et al. (2000) J. Biol. Chem. 275:6733-6738). In earlier studies by this group, stimulation of CD4+ cells did not necessarily depend on the presence of APCs, and the mitogenic properties of these molecules on T cells derived from rat and mouse species was different: rat splenocytes proliferated in response to CP1 treatment, while mouse splencocytes did not (Tzianabos et al. (1995) J. Clin. Invest. 96:2727-2731; Brubaker et al. (1999) J. Immunol. 162:2235-2242).
Overall, however, their observations led this group to hypothesize that the activation of CD4+ T cells by these polysaccharides leads to the production of cytokines such as IL2 or IL10 that protect against inflammatory responses (PCT International Publication WO 00/59515; Kalka-Moll et al. (2000) J. Immunol. 164:719-724; Tzianabos et al. (2000) J. Biol. Chem. 275:6733-6738; Tzianabos et al. (1999) J. Immunol. 163: 893-897). It remains unclear, however, exactly how these molecules activate T cells or how they exert their protective effects. Further confounding an understanding of these polysaccharides, this group has reported other studies indicating that the same zwitterionic polysaccharides can induce the formation of abscesses in the same in vivo model where protective effects of these molecules have been observed (Tzianabos et al. (1993) Science 262: 416-419; Tzianabos et al. (1994) Infect. Immun. 62:3590-3593). Therefore, from this body of literature, it is difficult to ascertain the mechanism whereby these zwitterionic polysaccharides act as suppressive modulators of the immune system in vivo.
Another group of investigators has described immunomodulatory effects of the exopolysaccharide (capsule-like) of Paenibacillus jamilae, a gram positive bacillus isolated from olive mill wastewaters (Ruiz-Bravo et al. (2001) Clin. Diag. Lab. Immunol. 8:706-710). Although the authors do not disclose the structural features of this polysaccharide, their results are similar to the work of Kasper and Tzianabos, summarized above. The molecule, referred to as CP-7, stimulates the proliferation of lymphocytes in culture, as well as significant expression of IFNγ and GMCSF. Further, this group reports that this compound renders mice resistant to Listeria monocytogenes infection. The investigators suggest that the mechanism may be through the stimulation of an inflammatory Th1 response.
From the body of research discussed above, one can conclude that a poly-saccharide antigen may induce a pro-inflammatory or anti-inflammatory response depending on structural features which are not presently fully understood.
In view of the confusing and sometimes contradictory effects reported in the literature for various immunomodulatory polysaccharides, there exists a need in the art for an understanding of the structural bases underlying immunomodulation by polysaccharides, pro-inflammatory as well as anti-inflammatory. There exists as well as a need for additional therapeutic molecules, both pro-inflammatory and anti-inflammatory, that modulate the immune response in a safe and effective manner. Such insight and additional molecules will facilitate the development of even more effective immunotherapeutic strategies for disease prevention and treatment.